Heart disease is one of the leading causes of death in newborn and in adults. Efforts to promote cardiac repair through use of stem cells hold promise but have been limited by technical considerations. The candidate recently found that the protein thymosin [unreadable]4 promotes cardiac cell migration in the embryonic heart and retains this property in neonatal heart muscle cells (cardiomyocytes). Survival of embryonic and neonatal cardiomyocytes in culture was also enhanced by thymosin [unreadable]4. After coronary artery ligation in mice, intraperitoneal or intracardiac administration of thymosin [unreadable]4 resulted in an activation of cell survival pathways, enhanced early myocyte survival and improved cardiac function. However, the exact mechanism by which thymosin [unreadable]4 achieves the observed effects is unknown. The hypotheses are that (1) thymosin [unreadable]4 promotes cardiomyocyte survival and repair through a mechanism similar to that induced by short hypoxic episodes in a process called ischemic preconditioning; (2) the variable N or C terminal domains of thymosin [unreadable]4 are responsible for the observed cardiac effects; and (3) that secreted thymosin [unreadable]4 enters cells by a receptor-mediated process. [unreadable] [unreadable] To address these issues, Specific Aim 1 will determine the gene and protein expression changes induced by thymosin [unreadable]4 in a low oxygen state, in which the regulation of several markers of altered metabolism in oxygen-starved tissue will be examined with or without thymosin [unreadable]4 treatment by microarray, Western blot, and histochemistry. Specific Aim 2 will determine specific domain(s) of thymosin [unreadable]4 responsible for myocardial cell migration, survival, and cardiac regeneration, in which fragments or biochemically modified forms of Thymosin [unreadable]-4 will be synthesized to test the biological function in vitro on embryonic cardiac explants and in vivo on mouse models of myocardial infarction. Specific Aim 3 will determine how cells internalize secreted thymosin [unreadable]4 from the extracellular environment, in which internalization of thymosin [unreadable]4 will be studied by live-cell imaging technology, and direct protein interactions will be studied by phage display and confirmed biochemically, histologically, and functionally. [unreadable] [unreadable] [unreadable]